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Despite the wide applications, systematic mechanobiological investigation of 3D porous scaffolds has yet to be performed due to the lack of methodologies for decoupling the complex interplay between structural and mechanical properties. Here, we discover the regulatory effect of cryoprotectants on ice crystal growth and use this property to realize separate control of the scaffold pore size and stiffness. Fibroblasts and macrophages are sensitive to both structural and mechanical properties of the gelatin scaffolds, particularly to pore sizes. Interestingly, macrophages within smaller and softer pores exhibit pro-inflammatory phenotype, whereas anti-inflammatory phenotype is induced by larger and stiffer pores. The structure-regulated cellular mechano-responsiveness is attributed to the physical confinement caused by pores or osmotic pressure. Finally, in vivo stimulation of endogenous fibroblasts and macrophages by implanted scaffolds produce mechano-responses similar to the corresponding cells in vitro, indicating that the physical properties of scaffolds can be leveraged to modulate tissue regeneration.

Cryoprotectant enables structural control of porous scaffolds for exploration of cellular mechano-responsiveness in 3D